Compositions and methods for detecting a neoplasia

ABSTRACT

The invention provides compositions and methods for detecting a neoplasia (e.g., pancreatic cancer, lung cancer, colon cancer) in a subject sample (e.g., serum, blood, plasma, tissue). In particular embodiments, the invention provides methods for detecting BNC1 and ADAMTS1 promoter methylation in circulating DNA in serum.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No.13/699,452, filed on Jul. 3, 2013, now U.S. Pat. No. 10,167,513, whichis the 35 U.S.C. § 371 U.S. national stage entry of InternationalApplication PCT/US2011/037926 (WO 2011/150075) having an Internationalfiling date of May 25, 2011 which claims benefit of the following U.S.Provisional Application No. 61/348,010, filed May 25, 2010, the entirecontents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grants from the NationalInstitutes of Health, Grant No. NIH K23CA127141The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 1, 2019, isnamed 048317-410D01US.txt and is 24,000 bytes in size.

BACKGROUND OF THE INVENTION

Pancreatic cancer is the fourth leading cause of cancer mortality with adeath rate nearly equal to the incidence of this disease. Detection ofcancer specific, abnormally DNA methylated gene promoter sequences hasemerged as one of the leading tumor biomarker detection strategies.Recently, there have been successful reports of DNA methylationscreening using various body fluids, such as stool for detection ofcolorectal cancer, sputum for lung cancer, and urine for prostatecancer.

However, in pancreatic cancer no screening tool is currently availablefor early detection. This is particularly relevant as pancreatic canceris often found once it is already metastatic or locally advanced and thediagnosis is often delayed because patients present with nonspecificgastrointestinal symptoms. The development of a screening modality forpancreatic cancer which identifies early stage cancers amenable tosurgical curative resection would then have a potential impact inreducing mortality from this currently lethal disease.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods for diagnosing neoplasia (e.g., pancreatic cancer, colon cancer,lung cancer) featuring BNC1 and ADAMTS1. Advantageously, the methodprovides for screening serum for increased promoter methylation of BNC1and ADAMTS1 to identify early stage neoplasia or a propensity to developa neoplasia.

In one aspect, the invention provides a method for detecting orcharacterizing a neoplasia in a biologic sample of a subject, the methodinvolving detecting the methylation of a BNC1 and/or ADAMTS1 gene, wheredetection of methylation detects or characterizes the presence of aneoplasia in the sample. In one embodiment, the method detects anincrease in methylation relative to a reference. In another embodiment,the method detects promoter methylation or methylation of exon 1.

In another aspect, the invention provides a method for detecting orcharacterizing lung or colon cancer in a sample derived from a subject,the method involving detecting methylation of a BNC1 and ADAMTS1 gene,where detection of methylation detects or characterizes lung or coloncancer in the subject. In one embodiment, the method involves detectingan alteration in the sequence or expression level of a Brca1, Brca2,p16, K-ras, APC, EGFR, and/or EML-ALK4 gene or polypeptide. In anotherembodiment, the subject is identified as having a propensity to developa neoplasia (e.g., is identified as a smoker, having colon polyps oradenomas, or a family history of cancer).

In another aspect, the invention provides a method for detecting orcharacterizing pancreatic cancer in a serum or plasma sample derivedfrom a subject, the method involving detecting the methylation of BNC1and ADAMTS1, where detection of methylation detects or characterizespancreatic cancer in the subject. In one embodiment, the method detectsan increase in methylation relative to a reference. In anotherembodiment, the method further involves imaging the subject, andlocalizing the cancer. In another embodiment, the method furtherinvolves detecting an alteration in the sequence or expression of aBrca1, Brca2, p16, K-ras, APC, PalB2, and/or DPC4 gene or polypeptiderelative to a reference. In one embodiment, the subject is identified ashaving a propensity to develop a pancreatic cancer (e.g., is identifiedas a smoker, has a Brca1 or Brca2 mutation, pancreatic cyst, chronicpancreatitis, or a family history of cancer).

In another aspect, the invention provides a method of monitoring asubject diagnosed as having a neoplasia, the method involving detectingan alteration in promoter methylation level in a BNC1 and/or ADAMTS1gene in a subject sample relative to a reference, where an altered levelindicates an altered severity of neoplasia in the subject. In oneembodiment, the reference is the level of methylation present in asample previously obtained from the subject; is a baseline level ofmethylation present in a sample from the subject obtained prior totherapy; or is the level of methylation present in a normal patientsample. In another embodiment, a decreased level of methylation relativeto a reference indicates a reduced severity of the neoplasia, and anincreased level of methylation relative to a reference indicates anincreased severity of neoplasia.

In yet another aspect, the invention provides a method for selecting atreatment for a subject diagnosed as having a neoplasia, the methodinvolving detecting methylation of a BNC1 and/or ADAMTS1 gene, wheredetection of methylation indicates that epigenetic therapy should beselected for treatment of said subject. In one embodiment, theepigenetic therapy is selected from the group consisting of entinostat,SAHA (suberoylanilide hydroxamic acid), depsipeptide, azocytidine, anddeazocytidine.

In another aspect, the invention provides a kit for the analysis ofpromoter methylation, the kit involving at least one primer capable ofdistinguishing between methylated and unmethylated BNC1 and ADAMTS1promoter sequences. In one embodiment, the kit further contains a pairof primers for amplifying the promoter sequence of a reference gene. Inanother embodiment, the kit further contains a detectable probe, wherethe probe is capable of binding to the promoter sequence. In yet anotherembodiment, the probe is detected by fluorescence, by autoradiography,by an immunoassay, by an enzymatic assay, or by a colorimetric assay.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the neoplasia is a cancer that ispancreatic cancer, gastrointestinal cancer, lung cancer, colon cancer,duodenal cancer, colorectal carcinoma, neuroendocrine carcinoma,cholangiocarcinomas, or ampullary tumors. In other embodiments of theabove aspects, the biologic sample is a tissue or biologic fluid sample(e.g., of blood, serum, plasma, urine, pancreatic juice, pancreatic cystfluid, or lung lavage). In various embodiments, the reference is thelevel of methylation present at the promoter in a control sample. Inother embodiments, the control sample is derived from a healthy subject.In still other embodiments, the methylation is detected by quantitativemethylation-specific PCR (QMSP). In other embodiments, the level ofmethylation is quantified or the frequency of methylation is quantified.In still other embodiments, the methylation levels of the BNC1 andADAMTS1 promoters are quantified. In still other embodiments, the methodresults in at least 50%-100% (e.g., 50%, 60%, 70%, 80%, 90%, 100%)sensitivity. In still other embodiments, the subject is identified ashaving a propensity to develop a neoplasia (e.g., is a smoker, has aBrca1 or Brca2 mutation, pancreatic cyst, chronic pancreatitis, presenceof colon polyps or adenomas, or a family history of cancer). In variousembodiments, the method further involves detecting an alteration in thesequence or expression level of a Brca1, Brca2, p16, K-ras, APC, PalB2,DPC4, EGFR, and/or EML-ALK4 gene or polypeptide. In still otherembodiments, the alteration is a sequence alteration or alteration inexpression level. In still other embodiments, the subject is a humanpatient. In various embodiments of the above aspects, the methylation isdetected or quantified by methylation on beads or quantitativemethylation-specific PCR.

The invention provides compositions and methods for detecting promotermethylation of BNC1 and ADAMTS1 genes. Compositions and articles definedby the invention were isolated or otherwise manufactured in connectionwith the examples provided below. Other features and advantages of theinvention will be apparent from the detailed description, and from theclaims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “BNC1 gene” is meant a polynucleotide encoding a BNC1 polypeptide. Anexemplary BNC1 gene nucleotide sequence is publically available atGeneBank No.: NM_001717. An exemplary sequence is provided below.

   1 gcggcggggg cggccatcgt gctgcgcagc ctgggcgctt ggggagccgc ccacttcgcc  61 gggtcgcgcc ccgacggccg gagcgtggat gcggcggcgc ccgccgagcc ggggcggacg 121 cggggcggcc cgggcccggg agacgcgccg gcagccccgg caccgcagcg gtcgcaggat 181 ggccgaggct atcagctgta ctctgaactg tagttgccaa agtttcaaac ccgggaaaat 241 aaaccaccgt cagtgtgacc aatgcaagca tggatgggtg gcccacgctc taagtaagct 301 aaggatcccc cccatgtatc caacaagcca ggtggagatt gtccagtcca atgtagtgtt 361 tgatattagc agcctcatgc tctatgggac ccaggccatc cccgttcgcc taaaaatcct 421 actggaccgg ctcttcagtg tgttgaagca agatgaggtt ctccagatcc tccatgcctt 481 ggactggaca cttcaggatt atatccgtgg atacgtactg caggatgcat caggaaaggt 541 gttggatcac tggagcatca tgaccagtga ggaagaagtg gccaccttgc agcagttcct 601 tcgttttgga gagaccaaat ctatagttga actcatggca attcaagaga aagaagagca 661 atccatcatc ataccacctt ccacagcaaa tgtagatatc agggctttca tcgagagctg 721 cagtcacagg agttctagcc tccccactcc tgtggacaaa ggaaacccca gcagtataca 781 cccctttgag aacctcataa gcaacatgac tttcatgctg cctttccagt tcttcaaccc 841 tctgcctcct gcactgatag ggtcattgcc cgaacaatat atgttggagc agggtcatga 901 ccaaagtcag gaccccaaac aggaagtcca tgggcccttc cctgacagca gcttcttaac 961 ttccagttcc acaccatttc aggttgaaaa agatcagtgt ttaaactgtc cggatgctat1021 tactaaaaaa gaagacagca cccatttaag tgactccagc tcatacaaca ttgtcactaa1081 gtttgaaagg acacagttat cccctgaggc caaagtgaag cctgagagga atagccttgg1141 tacaaagaag ggccgggtgt tctgcactgc atgtgagaag accttctatg acaaaggcac1201 cctcaaaatc cactacaatg ccgtccactt gaagatcaag cataagtgca ccatcgaagg1261 gtgtaacatg gtgttcagct ccctaaggag ccggaatcgc catagcgcca accccaaccc1321 tcggctgcac atgccaatga acagaaataa ccgggacaaa gacctcagga acagcctgaa1381 cctggccagc tctgagaact acaagtgccc aggtttcaca gtgacgtccc cagactgtag1441 gcctcctccc agctaccctg gttcaggaga ggattccaaa ggccaaccag ccttcccaaa1501 cattgggcaa aatggtgtgc tttttcccaa cctaaagaca gtccagccag tccttccttt1561 ctaccgcagt ccagccacgc ctgccgaggt agcaaacacg cctgggatac tcccttccct1621 cccgctgttg tcctcttcaa tcccagaaca gctcatttca aacgaaatgc catttgatgc1681 ccttcccaag aagaaatcca ggaagtccag tatgcctatc aaaatagaga aagaagctgt1741 ggaaatagct aatgagaaaa gacacaacct cagctcagat gaagacatgc ccctacaggt1801 ggtcagtgaa gatgagcagg aggcctgcag tcctcagtca cacagagtat ctgaggagca1861 gcatgtacag tcaggaggct tagggaagcc tttccctgaa ggggagaggc cctgccatcg1921 tgaatcagta attgagtcca gtggagccat cagccaaacc cctgagcagg ccacacacaa1981 ttcagagagg gagactgagc agacaccagc attgatcatg gtgccaaggg aggtcgagga2041 tggtggccat gaacactact tcacacctgg gatggaaccc caagttcctt tttctgacta2101 catggaactg cagcagcgcc tgctggctgg gggactcttc agtgctttgt ccaacagggg2161 aatggctttt ccttgtcttg aagattctaa agaactggag cacgtgggtc agcatgcatt2221 agcaaggcag atagaagaaa atcgcttcca gtgtgacatc tgcaagaaga cctttaaaaa2281 tgcttgtagt gtgaaaattc atcacaagaa tatgcatgtc aaagaaatgc acacatgcac2341 agtggagggc tgtaatgcta cctttccctc ccgcaggagc agagacagac acagctcaaa2401 cctaaacctc caccaaaaag cattgagcca ggaagcattg gagagtagtg aagatcattt2461 ccgtgcagct taccttctga aagatgtggc taaggaagcc tatcaggatg tggcttttac2521 acagcaagcc tcccagacat ctgtcatctt caaaggaaca agtcgaatgg gcagtctggt2581 ttacccaata acgcaagtcc acagtgccag cctggagagc tacaactctg gccccttgag2641 cgagggcacc atcctggatt tgagcactac ctcgagcatg aagtcagaga gtagcagcca2701 ttcttcctgg gactctgacg gggtgagtga ggaaggcact gtgcttatgg aggacagtga2761 tgggaactgt gaagggtcga gccttgtccc tggggaagat gagtacccca tctgtgtcct2821 gatggagaag gctgaccaga gccttgctag cctgccttct gggttgccca taacctgtca2881 tctctgccaa aagacataca gtaacaaagg gacctttagg gcccactaca aaactgtgca2941 cctccggcag ctccacaaat gcaaagtacc aggctgcaac accatgtttt cgtctgttcg3001 cagtcgaaac agacacagcc agaatcccaa cctgcacaaa agcctggcct catctccaag3061 tcacctccag taacaagatg gcaaaccaag tatgctcaga taagcttttt tcataattca3121 ggaataaagt agtccataga aatgtttctg tttcatatca tttggggcga gtcaggcaaa3181 agtatttgat ttgactttat agttttccac agcacaatga gcaaaagaca aacctcgtgg3241 gaagatgaca ctggggcagc ccttcctatt atttttctta gcccaagagg tctttcactg3301 atacaaggaa aacttgcaga aatgtgattt ttcccagatt tgtttacatg ttccctggga3361 cagatccagg tctgcagatc gacaccagtg ggcccaggac ctgggggtgg ctttaaatga3421 ggcttgcagt gttaaaggtc ttggataaga agggtcctgg ggaagaagac tctgtggaca3481 agataccagt ccccaaaaca gcattttcag ttccttcttc aattagtttg aaatccagac3541 ctgagtttgg aagactgatt ttttgagacc atccctgtgt ttggagtgga taattgtccc3601 tcccctcagc cctgcaccag aggtctcata tgttacccca gggagttctc agaggattgg3661 gttggcctct aacatgttcc ttgttaattc ttgttctgta acatgcattc aagaagctag3721 gggaaaaata tctcatgcac ttaaataatg gtcttcaatt taatttaaaa atattttgac3781 aatatttaat ttgtgcttat gtggtgtttg gtgtgagtgc agatattgca ctgtgtcacc3841 tctggatctc tgctcagaag cagaacaagt gatgacctaa atgtcaaaat cactgctcgt3901 tttcatttgg tgaacttcaa actctgttct ttttggtcac ctgtggaatg aatgcaagca3961 tgattttggc aggaacattt gtacatattc tgccgtagat aatgtggttc tgatggttgt4021 tgtgtatttt cagtatcact ggatccctca gtcttcaccg ttttataaac gtataagatt4081 aggatgaact tttgaattta cttggtagga aaaaaagtag gacattattg ccatattgta4141 tgtcttaata tttaacttat tcggaaatat attccacact gttacataca ttttccatgg4201 tagaaaggaa gttcagtcag tcctgtggaa tgaaaccatc tcctaaaatt cagcatttgc4261 agcattctaa aagcctgtgt aggtacaagg acattgattt tgtattcaga attcaagtta4321 actatctttt aaattcgtgg ttgatgtaag taataaaaaa cattcttaaa gttgagggtt4381 ataagagaga ttatttctgt ggtctaaagg ttaaaaagcc aacaacctgt taccaattat4441 ttcagctttt tttgttttaa taagtgtgac aacttaaaac ttgtttctat ttaaagtgaa4501 atgtatcttt caactgttta gttacccagc tgtttaatat tccagtcttc ccaaagtgaa4561 aagatttgta tacaaatgtt ttctatgatt taataaaaat atatggcaca ccaaaaaaaa4621 aaaaaaa

By “BNC1 promoter” is meant a polynucleotide sequence sufficient todirect expression of a BNC1 coding sequence. The sequence of anexemplary BNC1 promoter (BNC1 at chr15:81715659-81744472) is providedbelow:

BNC1 promoter region upstream 1 kb from Exon 1gtttccttttcaaggatccccaccagcagcaaggcaggtggaagaaactgagggaagaggaagcaggacccccgccgcccccccagtctagcctttacagactccttttaaggagctgaagaggttgggagaggcttgccctgccccggggcagcagaaacccgggcgctgctgcagcagctgtgggcgatctccgtcagctcctgggctccaggatcctgcggcctcagcccctccgcgtctactctcggggccaccacctccagttggcttctccagggcacttcatttcattccgtccctagcatccaataagacgcgaaaaacttaatcccattttacaactgaagccactgaggcccagagagcaaagccatttgtcctgggaaagtggtcaagctgggactttgactcttcagaccacccaagggaaggggtgtgtgtgtgtgtgtgtgtgtgtgtgcgtgtgcggtggggggcgcttgttttgagttcttaagaaaacctcctggcgacccccttcttccacatcccaagacgctcgtcccgcactttctcgggaatgaggtttctgcaggcgagggcggcgctgccttcttcctccgcggcagtgagaccccgagggcgccccagggtaggaggggaggccgaatcatctcctgagaagagcgccagagaacttcagagcgtttcgcccttccccgggagaggcaaacaccgacacgtctgtgtcttttaccaacaagtgccttcaagcccggcgggggcagacacctccgcgccggccgccggcgaggtctccgcggtctgcgggggccacggcctcgcctcagctgcgctgatttagggcgttatccggtcccggggcgggaggcggcctcccgggcggcgaagcagcgcccgcggcgtggggcgaccgcgcggtgggcggaggggcagggggaggggcggagaggcgtccccggggcgcagggggcgggcgtgcgggcacacgcggtgc BNC1 Exon 1GCGGCGGGGGCGGCCATCGTGCTGCGCAGCCTGGGCGCTTGGGGAGCCGCCCACTTCGCCGGGTCGCGCCCCGACGGCCGGAGCGTGGATGCGGCGGCGCCCGCCGAGCCGGGGCGGACGCGGGGCGGCCCGGGCCCGGGAGACGCGCCGGCAGCCCCGGCACCGCAGCGGTCGCAGGATGGCCGAGgtaagcgcggcgc

By “ADAMTS1 gene” is meant a polynucleotide sequence encoding a ADAMTS1protein. An exemplary ADAMTS1 gene sequence is publically available atGeneBank No.: NM_006988. An exemplary ADAMTS1 gene sequence is providedbelow:

   1 gcactcgctg gaaagcggct ccgagccagg ggctattgca aagccagggt gcgctaccgg  61 acggagaggg gagagccctg agcagagtga gcaacatcgc agccaaggcg gaggccgaag 121 aggggcgcca ggcaccaatc tccgcgttgc ctcagccccg gaggcgcccc agagcgcttc 181 ttgtcccagc agagccactc tgcctgcgcc tgcctctcag tgtctccaac tttgcgctgg 241 aagaaaaact tcccgcgcgc cggcagaact gcagcgcctc cttttagtga ctccgggagc 301 ttcggctgta gccggctctg cgcgcccttc caacgaataa tagaaattgt taattttaac 361 aatccagagc aggccaacga ggctttgctc tcccgacccg aactaaaggt ccctcgctcc 421 gtgcgctgct acgagcggtg tctcctgggg ctccaatgca gcgagctgtg cccgaggggt 481 tcggaaggcg caagctgggc agcgacatgg ggaacgcgga gcgggctccg gggtctcgga 541 gctttgggcc cgtacccacg ctgctgctgc tcgccgcggc gctactggcc gtgtcggacg 601 cactcgggcg cccctccgag gaggacgagg agctagtggt gccggagctg gagcgcgccc 661 cgggacacgg gaccacgcgc ctccgcctgc acgcctttga ccagcagctg gatctggagc 721 tgcggcccga cagcagcttt ttggcgcccg gcttcacgct ccagaacgtg gggcgcaaat 781 ccgggtccga gacgccgctt ccggaaaccg acctggcgca ctgcttctac tccggcaccg 841 tgaatggcga tcccagctcg gctgccgccc tcagcctctg cgagggcgtg cgcggcgcct 901 tctacctgct gggggaggcg tatttcatcc agccgctgcc cgccgccagc gagcgcctcg 961 ccaccgccgc cccaggggag aagccgccgg caccactaca gttccacctc ctgcggcgga1021 atcggcaggg cgacgtcggc ggcacgtgcg gggtcgtgga cgacgagccc cggccgactg1081 ggaaagcgga gaccgaagac gaggacgaag ggactgaggg cgaggacgaa ggggctcagt1141 ggtcgccgca ggacccggca ctgcaaggcg taggacagcc cacaggaact ggaagcataa1201 gaaagaagcg atttgtgtcc agtcaccgct atgtggaaac catgcttgtg gcagaccagt1261 cgatggcaga attccacggc agtggtctaa agcattacct tctcacgttg ttttcggtgg1321 cagccagatt gtacaaacac cccagcattc gtaattcagt tagcctggtg gtggtgaaga1381 tcttggtcat ccacgatgaa cagaaggggc cggaagtgac ctccaatgct gccctcactc1441 tgcggaactt ttgcaactgg cagaagcagc acaacccacc cagtgaccgg gatgcagagc1501 actatgacac agcaattctt ttcaccagac aggacttgtg tgggtcccag acatgtgata1561 ctcttgggat ggctgatgtt ggaactgtgt gtgatccgag cagaagctgc tccgtcatag1621 aagatgatgg tttacaagct gccttcacca cagcccatga attaggccac gtgtttaaca1681 tgccacatga tgatgcaaag cagtgtgcca gccttaatgg tgtgaaccag gattcccaca1741 tgatggcgtc aatgctttcc aacctggacc acagccagcc ttggtctcct tgcagtgcct1801 acatgattac atcatttctg gataatggtc atggggaatg tttgatggac aagcctcaga1861 atcccataca gctcccaggc gatctccctg gcacctcgta cgatgccaac cggcagtgcc1921 agtttacatt tggggaggac tccaaacact gccccgatgc agccagcaca tgtagcacct1981 tgtggtgtac cggcacctct ggtggggtgc tggtgtgtca aaccaaacac ttcccgtggg2041 cggatggcac cagctgtgga gaagggaaat ggtgtatcaa cggcaagtgt gtgaacaaaa2101 ccgacagaaa gcattttgat acgccttttc atggaagctg gggaatgtgg gggccttggg2161 gagactgttc gagaacgtgc ggtggaggag tccagtacac gatgagggaa tgtgacaacc2221 cagtcccaaa gaatggaggg aagtactgtg aaggcaaacg agtgcgctac agatcctgta2281 accttgagga ctgtccagac aataatggaa aaacctttag agaggaacaa tgtgaagcac2341 acaacgagtt ttcaaaagct tcctttggga gtgggcctgc ggtggaatgg attcccaagt2401 acgctggcgt ctcaccaaag gacaggtgca agctcatctg ccaagccaaa ggcattggct2461 acttcttcgt tttgcagccc aaggttgtag atggtactcc atgtagccca gattccacct2521 ctgtctgtgt gcaaggacag tgtgtaaaag ctggttgtga tcgcatcata gactccaaaa2581 agaagtttga taaatgtggt gtttgcgggg gaaatggatc tacttgtaaa aaaatatcag2641 gatcagttac tagtgcaaaa cctggatatc atgatatcat cacaattcca actggagcca2701 ccaacatcga agtgaaacag cggaaccaga ggggatccag gaacaatggc agctttcttg2761 ccatcaaagc tgctgatggc acatatattc ttaatggtga ctacactttg tccaccttag2821 agcaagacat tatgtacaaa ggtgttgtct tgaggtacag cggctcctct gcggcattgg2881 aaagaattcg cagctttagc cctctcaaag agcccttgac catccaggtt cttactgtgg2941 gcaatgccct tcgacctaaa attaaataca cctacttcgt aaagaagaag aaggaatctt3001 tcaatgctat ccccactttt tcagcatggg tcattgaaga gtggggcgaa tgttctaagt3061 catgtgaatt gggttggcag agaagactgg tagaatgccg agacattaat ggacagcctg3121 cttccgagtg tgcaaaggaa gtgaagccag ccagcaccag accttgtgca gaccatccct3181 gcccccagtg gcagctgggg gagtggtcat catgttctaa gacctgtggg aagggttaca3241 aaaaaagaag cttgaagtgt ctgtcccatg atggaggggt gttatctcat gagagctgtg3301 atcctttaaa gaaacctaaa catttcatag acttttgcac aatggcagaa tgcagttaag3361 tggtttaagt ggtgttagct ttgagggcaa ggcaaagtga ggaagggctg gtgcagggaa3421 agcaagaagg ctggagggat ccagcgtatc ttgccagtaa ccagtgaggt gtatcagtaa3481 ggtgggatta tgggggtaga tagaaaagga gttgaatcat cagagtaaac tgccagttgc3541 aaatttgata ggatagttag tgaggattat taacctctga gcagtgatat agcataataa3601 agccccgggc attattatta ttatttcttt tgttacatct attacaagtt tagaaaaaac3661 aaagcaattg tcaaaaaaag ttagaactat tacaacccct gtttcctggt acttatcaaa3721 tacttagtat catgggggtt gggaaatgaa aagtaggaga aaagtgagat tttactaaga3781 cctgttttac tttacctcac taacaatggg gggagaaagg agtacaaata ggatctttga3841 ccagcactgt ttatggctgc tatggtttca gagaatgttt atacattatt tctaccgaga3901 attaaaactt cagattgttc aacatgagag aaaggctcag caacgtgaaa taacgcaaat3961 ggcttcctct ttcctttttt ggaccatctc agtctttatt tgtgtaattc attttgagga4021 aaaaacaact ccatgtattt attcaagtgc attaaagtct acaatggaaa aaaagcagtg4081 aagcattaga tgctggtaaa agctagagga gacacaatga gcttagtacc tccaacttcc4141 tttctttcct accatgtaac cctgctttgg gaatatggat gtaaagaagt aacttgtgtc4201 tcatgaaaat cagtacaatc acacaaggag gatgaaacgc cggaacaaaa atgaggtgtg4261 tagaacaggg tcccacaggt ttggggacat tgagatcact tgtcttgtgg tggggaggct4321 gctgaggggt agcaggtcca tctccagcag ctggtccaac agtcgtatcc tggtgaatgt4381 ctgttcagct cttctgtgag aatatgattt tttccatatg tatatagtaa aatatgttac4441 tataaattac atgtacttta taagtattgg tttgggtgtt ccttccaaga aggactatag4501 ttagtaataa atgcctataa taacatattt atttttatac atttatttct aatgaaaaaa4561 acttttaaat tatatcgctt ttgtggaagt gcatataaaa tagagtattt atacaatata4621 tgttactaga aataaaagaa cacttttgga aaaaaaaaaa aaaaaaaaaa

By “ADAMTS1 promoter” is meant is meant a polynucleotide sequencesufficient to direct expression of a BNC1 coding sequence. The sequenceof an exemplary AdamTS1 (ADAMTS1 at chr21:27130477-27139599) promoter isprovided below:

ADAMTS1 promoter region upstream 1 kb from Exon 1atttccttctttccccctctgcacgcttgctagccccagcgatcgctgctggcccccgggtaggaaagtggggttcctggccgtttctgcgacgctggcctagggcttgcagctgctgttgagtgaaagcacgcagactggcgggagccgatcatttctcgaatgaagaagaaaaagcgcaattccctccttatgctctagggaattgagccgcgtcccagatcacccattccagaaatgtgaaaccgggccctcacaaagtcgtctctggtgaagaggtggcgtgcggggtgggggttggtggagggtgaaggcataagcaaacatattttaaaatccagatcgtaggaagtgtcacctggcccctcacccaggcatgctttctgggggaagcgcagggccaagctttccctagaaaagctggggcgaagagagagcaggcggcggctaaggagctcctggcaggctgggaaggtggagaagtggggtgaggtatttttctagaaagtgtagccctagctcatctcctagattggggaagagggaactgagggaggagggaaggagacccagggcagctccaggatagggaaatgttgaagaagggactgcgttctccaaccgaaccctccctcctgggaaccgcagcccagcgcggtaactgagttaccgcaaccgggcggtggggaggaagggtggtccaggaaaccggcgagggagaaaagcggtggaagggagagtcttctccctggagcggccccagcagtacaaagtgctggtcacagcgccccttccgcccctagattgacgagcagtggcgtggagccagcgcggaggctgccccctccccctcccgagcccgcagcgcggagcgcggtttagcaccaacggagccgggggcggcgtctttgggatggaaaagggccaaaggggaggagtggggtgggggtgggggtttcactggtccactataaaaggaccgctcggctgcccggttctt ADAMTS1 Exon 1GCACTCGCTGGAAAGCGGCTCCGAGCCAGGGGCTATTGCAAAGCCAGGGTGCGCTACCGGACGGAGAGGGGAGAGCCCTGAGCAGAGTGAGCAACATCGCAGCCAAGGCGGAGGCCGAAGAGGGGCGCCAGGCACCAATCTCCGCGTTGCCTCAGCCCCGGAGGCGCCCCAGAGCGCTTCTTGTCCCAGCAGAGCCACTCTGCCTGCGCCTGCCTCTCAGTGTCTCCAACTTTGCGCTGGAAGAAAAACTTCCCGCGCGCCGGCAGAACTGCAGCGCCTCCTTTTAGTGACTCCGGGAGCTTCGGCTGTAGCCGGCTCTGCGCGCCCTTCCAACGAATAATAGAAATTGTTAATTTTAACAATCCAGAGCAGGCCAACGAGGCTTTGCTCTCCCGACCCGAACTAAAGGTCCCTCGCTCCGTGCGCTGCTACGAGCGGTGTCTCCTGGGGCTCCAATGCAGCGAGCTGTGCCCGAGGGGTTCGGAAGGCGCAAGCTGGGCAGCGACATGGGGAACGCGGAGCGGGCTCCGGGGTCTCGGAGCTTTGGGCCAGTACCCACGCTGCTGCTGCTCGCCGCGGCGCTACTGGCCGTGTCGGACGCACTCGGGCGCCCCTCCGAGGAGGACGAGGAGCTAGTGGTGCCGGAGCTGGAGCGCGCCCCGGGACACGGGACCACGCGCCTCCGCCTGCACGCCTTTGACCAGCAGCTGGATCTGGAGCTGCGGCCCGACAGCAGCTTTTTGGCGCCCGGCTTCACGCTCCAGAACGTGGGGCGCAAATCCGGGTCCGAGACGCCGCTT

By “alteration” is meant an increase or decrease. An alteration may beby as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%,or even by as much as 75%, 80%, 90%, or 100%. An alteration may be achange in sequence relative to a reference sequence or a change inexpression level, activity, or epigenetic marker (e.g., promotermethylation or histone alterations).

By “biologic sample” is meant any tissue, cell, fluid, or other materialderived from an organism.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “clinical aggressiveness” is meant the severity of the neoplasia.Aggressive neoplasias are more likely to metastasize than lessaggressive neoplasias. While conservative methods of treatment areappropriate for less aggressive neoplasias, more aggressive neoplasiasrequire more aggressive therapeutic regimens.

By “control” is meant a standard of comparison. For example, themethylation level present at a promoter in a neoplasia may be comparedto the level of methylation present at that promoter in a correspondingnormal tissue.

By “diagnostic” is meant any method that identifies the presence of apathologic condition or characterizes the nature of a pathologiccondition (e.g., a neoplasia). Diagnostic methods differ in theirsensitivity and specificity. While a particular diagnostic method maynot provide a definitive diagnosis of a condition, it suffices if themethod provides a positive indication that aids in diagnosis.

By “frequency of methylation” is meant the number of times a specificpromoter is methylated in a number of samples.

By “increased methylation” is meant a detectable positive change in thelevel, frequency, or amount of methylation. Such an increase may be by5%, 10%, 20%, 30%, or by as much as 40%, 50%, 60%, or even by as much as75%, 80%, 90%, or 100%. In certain embodiments, the detection of anymethylation in an AdamTS1 or BNC promoter in a subject sample issufficient to identify the subject as having a neoplasia, apre-cancerous lesion, or the propensity to develop a neoplasia.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By “methylation level” is meant the number of methylated alleles.Methylation level can be represented as the methylation present at atarget gene/reference gene×100. Any ratio that allows the skilledartisan to distinguish neoplastic tissue from normal tissue is useful inthe methods of the invention. One skilled in the art appreciates thatthe cutoff value is selected to optimize both the sensitivity and thespecificity of the assay. In certain embodiments, merely detectingpromoter methylation of BNC1 and AdamTS1 genes in a biological sample ofa subject is sufficient to identify the subject as having cancer, apre-cancerous lesion, or having a propensity to develop cancer.

By “tumor marker profile” is meant an alteration present in a subjectsample relative to a reference. In one embodiment, a tumor markerprofile includes promoter methylation of BNC1 and/or AdamTS1 genes, aswell as mutations present in Brca1, p16, K-ras, APC, PalB2, DPC4, EGFR,EML-ALK4 or other marker known in the art.

By “sensitivity” is meant the percentage of subjects with a particulardisease that are correctly detected as having the disease. For example,an assay that detects 98/100 of carcinomas has 98% sensitivity.

By “severity of neoplasia” is meant the degree of pathology. Theseverity of a neoplasia increases, for example, as the stage or grade ofthe neoplasia increases.

By “specificity” is meant the percentage of subjects without aparticular disease who test negative.

By “neoplasia” is meant any disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is an example of a neoplasia.Examples of cancers include, without limitation, pancreatic cancer,including islet cell and adenocarinomas), duodenal cancers,cholangiocarcinomas, ampullary tumors, leukemia's (e.g., acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colorectal carcinoma, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,neuroendocrine carcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

By “periodic” is meant at regular intervals. Periodic patient monitoringincludes, for example, a schedule of tests that are administered daily,bi-weekly, bi-monthly, monthly, bi-annually, or annually.

By “promoter” is meant a nucleic acid sequence sufficient to directtranscription. In general, a promoter includes, at least, 50, 75, 100,125, 150, 175, 200, 250, 300, 400, 500, 750, 1000, 1500, or 2000nucleotides upstream of a given coding sequence (e.g., upstream of thecoding sequence for BNC1 and ADAMTS1 polypeptides).

By “marker” is meant any protein or polynucleotide having an alterationin methylation, expression level or activity that is associated with adisease or disorder.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition. Exemplaryreferences include a baseline of methylation present in a healthycontrol subject or a standardized curve.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout ortherebetween.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show a schematic diagram, a gel, and a bar graph,respectively. FIG. 1A depicts a schematic that is a step-wiserepresentation of the screening method used to identify epigeneticbiomarkers. FIG. 1B depicts the results of methylation analysis of 8cancer-specific genes in pancreatic cancer cell lines. M=methylationsignal; U=unmethylated signal. IVD=in vitro methylated DNA. ddH₂O=watercontrol adding no DNA. FIG. 1C is a bar graph that displays themethylation frequencies of the 8 genes in a series of pancreatic cancerpatients from JHU (stage 1-4; n=143), along with a series of normalpancreas from non cancerous patients (n=4), and a series of pancreaticintraductal neoplasia (PanIN) (n=20 ranging from PanIN 1-3).

FIGS. 2A, 2B, and 2C depict promoter methylation gels and correspondingbar graphs, a CpG methylation diagram, and a schematic depictingmethylation-on-bead (MOB) technology, respectively. FIG. 2A showssilencing of BNC1 and ADAMTS1 genes in pancreatic cancer cell lines by amethylation specific PCR (MSP) analysis of BNC1 and ADAMTS1 genepromoter regions (upper panel depicting gel bands), and the correlationof promoter methylation with gene expression by quantitative RT-PCR) inpancreatic cancer cell lines (lower panel showing data in bar graphformat. M=methylation signal; U=unmethylated signal. IVD=in vitromethylated DNA. Real-time RT-PCR expression is shown as fold change ±SErelative to mock-treated cells during 5 uM 5-Aza-deoxycitidine (DAC) and300 nM Trichostatin A (TSA) treatments. FIG. 2B shows the results ofbisulfite sequencing analysis of CpG islands in the BNC1 gene promoterregion. The BNC1 promoter regions is depicted as a line, with CpGregions designated as vertical slashes. TIS indicates thetranscriptional start site. Location of CpG sites (bisulfite priners(BST): upstream region from −331 to +36 relative to transcriptionalstart site). Bisulfite sequencing analysis of the BNC1 gene in Panc1(pancreatic cancer cell line), normal pancreas tissue, pancreaticprimary tumors and HCT116 (DNMT1/3b−/−) as a negative control. Open andfilled circles represent unmethylated and methylated CpG sites,respectively, and each row represents a single clone. FIG. 2C showsstep-wise illustration of the nanoassay used to detect methylation inserum. DNA extraction and bisulfite conversion are carried out usingmethylation-on-beads (MOB). DNA is eluted using PCR buffer, andamplified using MSP modified primers and fluorophore-labeled NTPs.Samples are analyzed using MS-qFRET or gel electrophoresis.

FIG. 3 depicts plots showing the results of quantitative MSP (qMSP)analysis of BNC1 and ADAMTS1 using real-time PCR. FIG. 3 shows astatistically increased frequency of methylation when normal pancreastissue is compared to invasive cancers. FIG. 3 also shows that BNC1methylation could be detected at the earliest stages of pancreaticcarcinogenesis (e.g. PanIN's) as compared to ADAMTS1 methylation. Normalpancreas (n=4 normal tissues, n=10 surrounding normal tissues) andPancreatitis samples (n=30), PanIN (n=20), and tumor (stage 2; n=12),respectively. Real-time MSP is shown as fold change for Methylatedsignal relative to Unmethylated signal. In MSP analysis, signals forunmethylated (U) and methylated (M) DNA are shown for each sample.Horizontal bar indicates mean.

FIGS. 4A, 4B, and 4C show colony formation assays, Panc1 charts and bargraphs, and MIA-PaCa2 charts and bar graphs, respectively. FIG. 4A showscolony formation by Panc1 and MIA-PaCa2 cells transfected with pcDNA3.1or BNC1-pcDNA3.1 and grown for 2 weeks in medium containing G418.Results are plotted as the mean colony numbers relative to pcDNA3.1transfectants in three independent experiments (Panc1; *p=0.0275,MIA-PaCa2; *p=0.0294). FIG. 4B shows graphs that summarize cellproliferation assay in Panc1 cells, while FIG. 4C shows analogous datafor MIA-PaCa cells. FIGS. 4A and 4B are each divided into a right panel,a middle panel, and a left panel, which depict relevant data for Panc1and MIA-PaCa cells, respectively. Right panel: BNC1 transfected Panc1cells (BNC1-pcDNA3.1) were compared with control cells transfected withempty vector (pcDNA3.1). Results are plotted as the mean cells number inthree different independent experiments (Panc1; *p=0.0469; 72 hrs,**p=0.0086; 96 hrs: MIA-PaCa2, *p=0.0469; 48 hrs, *p=0.0318; 72 hrs,*p=0.0389; 96 hrs). Middle panel: Cell proliferation measured by3H-thymidine incorporation (Panc1; *p=0.0286, MIA-PaCa2; *p=0.0256).Left panel: Invasion of Panc1 and MIA-PaCa2 cells throughmatrigel-coated transwells relative to control cells transfected withempty vector in three independent experiments (NS: not statisticallysignificant).

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful foridentifying a subject as having or having a propensity to developneoplasia (e.g., pancreatic cancer, colon cancer, lung cancer).

The invention is based, at least in part, on the discovery thatdetection of BNC1 and ADAMTS1 promoter methylation was useful foridentifying with high sensitivity the earliest stages of pancreascancers. Notably, detection of BNC1 and ADAMTS1 promoter methylation incirculating DNA in serum was useful for the early detection of lung,colon and pancreatic cancer, especially in high risk individuals.Moreover, BNC1 is a tumor suppressor gene in pancreatic cancer that isinactivated by promoter methylation.

A genome-wide transcriptome approach was used to identify new cancerspecific DNA methylation alterations in pancreatic carcinoma.Methylation frequencies were analyzed for genes, BNC1 and ADAMTS1, bymethylation specific PCR and quantitative methylation specific PCR, aswell as expression analysis by real-time PCR and immunohistochemistry. Anovel nanoparticle-enabled Methylation On Beads technology was used todetect very early stage pancreatic cancers. The biological role of BNC1gene was examined by colony formation, cell proliferation, and invasionassays in pancreatic cancer cell lines. This analysis led to theidentification of BNC1 (91.8%) and ADAMTS1 (66.7%) as genes that showeda high frequency of methylation in pancreas cancer tissues (n=143). BNC1was frequently methylated in the earliest stages of pancreascarcinogenesis including carcinoma in situ or pancreatic intraepithelialneoplasia PanIN3 (100%) and Stage 1 invasive cancers (97.4%). Using theultrasensitive nanoparticle-enabled MOB assay, these alterations weredetected in serum samples from patients with pancreas cancer, with asensitivity for BNC1 of 79% (95% CI: 0.6-0.8) and for ADAMTS1 of 48%(95% CI: 0.3-0.6) (n=42 cancers, Stages 1-4), while specificity was 88%for BNC1 (95% CI: 0.6-0.9) and 92% for ADAMTS1 (95% CI: 0.7-0.9) among26 individuals without cancer. BNC1 overexpression in pancreatic cancercell lines showed suppressive effect by colony formation, and cellproliferation, but not invasion.

Pancreatic Cancer

Pancreatic cancer is a deadly cancer with an overall 5-year survivalrate of less than 5% and no improvements in survival over the last 3decades. Pancreatic cancer currently ranks as the fourth leading causeof cancer related death in United States with an estimated 42,470 newcases and 35,240 deaths in 2009 and its incidence is rising. One of themajor factors attributed to the dismal prognosis of pancreas cancer isthe delayed diagnosis of the disease. Only about 10% of cases areamenable to potential curative surgical resection. However, long term5-year survival is attainable in selected patients with early-stagepancreatic cancer who can undergo curative surgical resection. Earlydetection of pancreatic cancer is, therefore, thought to be the bestmodality for improving survival in this lethal disease. However, noscreening test is available for detection of pancreatic cancer.

Pancreatic cancer is characterized by multiple genetic and epigeneticchanges. In recent years, it has become apparent that pancreatic canceris as much a disease of mis-regulated epigenetics, as it is a disease ofgenetic mutation. In particular, changes in DNA promoter methylationpatterns likely play a crucial role in tumorigenesis and cancerprogression. In order to address the need for both clinical diagnosticsas well as therapeutics, many studies have employed DNA methylation ofspecific genes for application in diagnostics of multiple cancers. Suchdiagnostic tests can in principle be used for early detection ofcancers, for assessing prognosis, and for therapeutics as predictors ofresponse to therapy. Early detection of disease results in an improvedclinical outcome for most types of cancer (Jemal A et al. (2011) CancerStatistics, 2010. CA Cancer J Clin. March-April; 61(2):133-4).Therefore, much effort has been invested in developing efficientscreening technologies for early detection strategies.

Cancer Screening

Ideally, methods for cancer screening should be easy to perform,cost-effective, noninvasive, and provide a benefit to patients. Currentmethods of screening for pancreatic cancer are inadequate. For example,endoscopic ultrasound has shown promise for identifying high riskpatients, but requires access to specialized centers and is an expensiveand invasive modality that needs to be repeated at frequent intervals.The present invention provides significant advantages over existingscreening technologies for pancreatic and other cancers. Significantly,the invention provides methods for detecting BNC1 and ADAMTS1methylation. Increased methylation in BNC1 and ADAMTS1 is associatedwith all stages of pancreatic cancer. BNC1 gene was methylated at 100%frequency in PanIN3, which is felt to be the penultimate step prior todevelopment of invasive carcinoma.

Interestingly, BNC1 showed a potential tumor suppressive role asmeasured by suppression of colony formation, cell proliferation, andinvasion in pancreatic cancer cells. Methylation of BNC1 and ADAMTS1showed high sensitivity and specificity for detecting pancreatic cancerin sera using nano-enabled methylation based technology termedMethylation-on-Beads (MOB). The results reported herein below indicatethat methylation-based screening is useful for identifying subjects atrisk of not only pancreatic cancer, but also, lung and colon cancersusing a non-invasive inexpensive modality.

While the methods of the invention are suitable for screening thepopulation at large, it is particularly useful for screening subjectsidentified as at increased risk for having a neoplasia. Subjectsidentified as having an increased risk of neoplasia (e.g., pancreaticcancer, lung cancer, colon cancer) include but are not limited tosmokers and subjects having a Brca1 or Brca2 mutation. Subjectsidentified as having an increased risk of pancreatic cancer includesubjects identified as having pancreatic cysts or pancreatitis, as wellas patients that have a family history of pancreatic cancer,particularly in 1 or 2 relatives.

Types of Biological Samples

The level of promoter methylation in each of the genes identified herein(e.g., BNC1 and ADAMTS1) can be measured in different types of biologicsamples. In one embodiment, the biologic sample is a blood, plasma, orserum sample. In another embodiment, the sample is a tissue sample thatincludes cells of a tissue or organ (e.g., pancreatic cells, cells of apancreatic cyst or pancreas lesion, lung cells, and colon cells).Pancreatic tissue is obtained, for example, from a biopsy of thepancreas. In another embodiment, the biologic sample is a biologic fluidsample (e.g., blood, blood serum, plasma, urine, stool, pancreatic cystfluid, fluid from the major/minor pancreatic duct (i.e., “pancreaticjuice”) lung lavage, stool, sputum, or any other biological fluid usefulin the methods of the invention).

Methylation-On-Beads

In brief, Methylation-on-Beads is a single-tube method forpolynucleotide extraction and bisulfite conversion that provides a rapidand highly efficient method for DNA extraction, bisulfite treatment anddetection of DNA methylation using silica superparamagnetic particles(SSP). All steps are implemented without centrifugation or air dryingthat provides superior yields relative to conventional methods for DNAextraction and bisulfite conversion. SSP serve as solid substrate forDNA binding throughout the multiple stages of each process.Specifically, SSP are first used to capture genomic DNA from rawbiological samples, processed biological samples or cultured cells.Sodium bisulfite treatment is then carried out in the presence of SSPwithout tube transfers. Finally, the bisulfite treated DNA is analyzedto determine the methylation status. DNA extraction yield was found tobe 5-20 times the yield from conventional extraction. 90% of the inputDNA was recovered after bisulfite treatment. In addition,Methylation-on-Beads total process time was completed in less than 6hours when compared to 3 days for conventional methods. Hence,Methylation-on-Beads allows for convenient, efficient andcontamination-resistant methylation detection in a single tube or otherreaction platform. Methods for carrying out methylation-on-beads areknown in the art, and described, for example, in PCT/US2009/000039,which is incorporated herein in its entirety.

Methylation-Specific Quantum Dot FRET

If desired, methods of the invention may be advantageously combined withmethylation-specific quantum dot fluorescence resonance energy transfer(MS-qFRET). See, for example, PCT/US2009/000039, which is incorporatedherein in its entirety. MS-qFRET provides for the qualitative andquantitative detection of methylated DNA, as well as for the detectionof low-abundance methylated DNA. In this technique, quantum dots areused to capture methylation-specific PCR (MSP) amplicons and todetermine the methylation status via fluorescence resonance energytransfer (FRET). Desirably, MS-qFRET has low intrinsic background noise,high resolution and high sensitivity. MS-qFRET detects as little as 15pg of methylated DNA in the presence of a 10,000-fold excess ofunmethylated alleles, enables reduced use of PCR (8 cycles), and allowsfor multiplexed analyses.

More specifically, bisulfite-treated DNA is amplified through PCR,wherein the forward primer is biotinylated and the reverse primer islabeled with an organic fluorophore. Next, streptavidin-conjugatedquantum dots (QDs) are introduced to capture the labeled PCR productsvia streptavidin-biotin binding, bringing the QDs (serving as donors)and fluorophores (serving as acceptors) in close proximity allowing FRETto occur. Finally, PCR products are detected by emissions offluorophores accompanied by quenching of QDs. Spectral information isprocessed to determine the level of DNA methylation. Fluorescenceresponses are measured using a fluorospectrometer.

PCR with labeled primers is run. Products are then subject to PCRpurification (Qiagen Corporation) in order to recover PCR product thatis free of primers, primer-dimers, Taq and dNTPs. For conjugating withquantum dots (Invitrogen Corporation), 1 μL of 100 mM NaCl is mixed with7 μL PCR mix. 1 μL of deionized (DI) H₂0 is added to this mix. Finally,1 μL of 1 nM QD is added and the mixture is left undisturbed for 15minutes.

Mixtures of defined methylation levels ranging from 100%, 75%, 50%, 25%,and 1% of the total 150 ng input DNA are obtained. To quantify the levelof methylation, a “q-score”: a score that is based on the normalizedFRET efficiencies of acceptor and donor emission in MS-qFRET is defined.In any FRET process, as the level of the acceptor emission increases,the decay of donor emission increases as well. The FRET efficiency canthen be calculated based on the proximity ratio formalism,

$E = \frac{I_{A}}{I_{A} + I_{D}}$

(I_(D) and I_(A) corresponding to donor and acceptor intensity).Further, the q-score was determined by normalizing the calculated E forthe DNA mixture to an appropriate concentration of IVD only as amethylated control (q-score=1) and NL only and as an unmethylatedcontrol (q-score=0). By including positive and negative controls inevery assay a standard curve is created in order to quantify and comparemethylation levels of unknown samples using low-amplification cycles.

Diagnostic Assays

The present invention provides a number of diagnostic assays that areuseful for the identification or characterization of a neoplasia (e.g.,pancreatic cancer, lung cancer, colon cancer). In one embodiment, aneoplasia is characterized by quantifying or determining the methylationlevel of one or more of the following promoters: BNC1 and ADAMTS1 in theneoplasia. In one embodiment, methylation levels are determined usingquantitative methylation specific PCR (QMSP) to detect CpG methylationin genomic DNA. QMSP uses sodium bisulfate to convert unmethylatedcytosine to uracil. A comparison of sodium bisulfate treated anduntreated DNA provides for the detection of methylated cytosines.

While the examples provided below describe methods of detectingmethylation levels using QMSP, the skilled artisan appreciates that theinvention is not limited to such methods. Methylation levels arequantifiable by any standard method, such methods include, but are notlimited to real-time PCR, Southern blot, bisulfite genomic DNAsequencing, restriction enzyme-PCR, MSP (methylation-specific PCR),methylation-sensitive single nucleotide primer extension (MS-SNuPE)(see, for example, Kuppuswamy et al., Proc. Natl Acad. Sci. USA, 88,1143-1147, 1991), DNA microarray based on fluorescence or isotopelabeling (see, for example, Adorján Nucleic Acids Res., 30: e21 and HouClin. Biochem., 36:197-202, 2003), mass spectroscopy, methyl acceptingcapacity assays, and methylation specific antibody binding. See alsoU.S. Pat. Nos. 5,786,146, 6,017,704, 6,300,756, and 6,265,171.

The primers used in the invention for amplification of theCpG-containing nucleic acid in the specimen, after bisulfitemodification, specifically distinguish between untreated or unmodifiedDNA, methylated, and non-methylated DNA. Methylation specific primersfor the non-methylated DNA preferably have a T in the 3′ CG pair todistinguish it from the C retained in methylated DNA, and the complimentis designed for the antisense primer. Methylation specific primersusually contain relatively few Cs or Gs in the sequence since the Cswill be absent in the sense primer and the Gs absent in the antisenseprimer (C becomes modified to U (uracil) which is amplified as T(thymidine) in the amplification product).

The primers of the invention embrace oligonucleotides of sufficientlength and appropriate sequence so as to provide specific initiation ofpolymerization on a significant number of nucleic acids in thepolymorphic locus. Specifically, the term “primer” as used herein refersto a sequence comprising two or more deoxyribonucleotides orribonucleotides, preferably more than three, and most preferably morethan 8, which sequence is capable of initiating synthesis of a primerextension product, which is substantially complementary to a polymorphiclocus strand. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the inducing agentfor polymerization. The exact length of primer will depend on manyfactors, including temperature, buffer, and nucleotide composition. Theoligonucleotide primer typically contains between 12 and 27 or morenucleotides, although it may contain fewer nucleotides. Primers of theinvention are designed to be “substantially” complementary to eachstrand of the genomic locus to be amplified and include the appropriateG or C nucleotides as discussed above. This means that the primers mustbe sufficiently complementary to hybridize with their respective strandsunder conditions that allow the agent for polymerization to perform. Inother words, the primers should have sufficient complementarity with the5′ and 3′ flanking sequences to hybridize therewith and permitamplification of the genomic locus. While exemplary primers are providedherein, it is understood that any primer that hybridizes with the targetsequences of the invention are useful in the method of the invention fordetecting methylated nucleic acid.

In one embodiment, methylation specific primers amplify a desiredgenomic target using the polymerase chain reaction (PCR). The amplifiedproduct is then detected using standard methods known in the art. In oneembodiment, a PCR product (i.e., amplicon) or real-time PCR product isdetected by probe binding. In one embodiment, probe binding generates afluorescent signal, for example, by coupling a fluorogenic dye moleculeand a quencher moiety to the same or different oligonucleotidesubstrates (e.g., TaqMan® (Applied Biosystems, Foster City, Calif.,USA), Molecular Beacons (see, for example, Tyagi et al., NatureBiotechnology 14(3):303-8, 1996), Scorpions® (Molecular Probes Inc.,Eugene, Oreg., USA)). In another example, a PCR product is detected bythe binding of a fluorogenic dye that emits a fluorescent signal uponbinding (e.g., SYBR® Green (Molecular Probes)). Such detection methodsare useful for the detection of a methylation specific PCR product.

The methylation level of BNC1 and/or ADAMTS1 promoters described hereindefines the methylation profile of a neoplasia. The level of methylationpresent at any particular promoter is compared to a reference. In oneembodiment, the reference is the level of methylation present in acontrol sample obtained from a patient that does not have a neoplasia.In another embodiment, the reference is a baseline level of methylationpresent in a biologic sample derived from a patient prior to, during, orafter treatment for a neoplasia. In yet another embodiment, thereference is a standardized curve.

The methylation level of any one or more of the promoters describedherein (e.g., BNC1 and ADAMTS1) is used, alone or in combination withother standard methods, to characterize the neoplasia. For example,methods for detecting BNC1 or ADAMTS1 promoter methylation may becarried out prior to or concurrently with testing for alterations inother biomarkers, such as BRCA1, p16, K-ras, APC, PalB2, DPC4, EGFR,EML-ALK4 gene or polypeptide. In one embodiment, the alteration in aBrca1, p16, K-ras, APC, PalB2, DPC4, EGFR, EML-ALK4 gene is a mutationin the sequence of the gene relative to a reference. In otherembodiments, the alteration is in the level of expression or activity ofthe Brca1, p16, K-ras, APC, PalB2, DPC4, EGFR, or EML-ALK4 gene orpolypeptide. Once a subject is identified as having increased BNC1 orADAMTS1 promoter methylation, further diagnostic testing may be carriedout to localize the pathology to a particular organ or organ system.Typically, after the subject has been identified as having increasedpromoter methylation in BNC1 and ADAMTS1, imaging studies are carriedout. Such studies include, but are not limited to, endoscopicultrasound, MRI, CT scan, and PET scan.

If desired, diagnostic methods of the invention can be combined withconventional diagnostic methods used to determine the stage or grade ofa neoplasia. Grading is used to describe how abnormal or aggressive theneoplastic cells appear, while staging is used to describe the extent ofthe neoplasia. The grade and stage of the neoplasia is indicative of thepatient's long-term prognosis (i.e., probable response to treatment andsurvival). Thus, the methods of the invention are useful for predictinga patient's prognosis, and for selecting a course of treatment.

In conventional diagnostic methods, a pathologist will view a tissuesample from the tumor and determine the grade based on the degree ofpathology observed. Typically, pancreatic tumors are graded from 1-4.For a grade I tumor, cells present in the sample are most similar tonormal pancreatic cells. Grade 4 samples contain cells that are mostdissimilar to normal pancreatic cells. High-grade neoplasias are themost deadly because they are most aggressive and fast growing.High-grade neoplasias typically move rapidly into surrounding tissues,such as lymph nodes and bones.

Stage refers to the extent of a cancer. In pancreatic cancer, forexample, one staging method divides the cancer into four categories,stage I pancreatic cancer is found only in the pancreas itself or hasstarted to spread to the tissues next to the pancreas (such as the smallintestine, the stomach, or the bile duct). Stage II pancreatic cancerhas spread to nearby organs such as the stomach, spleen, or colon, buthas not entered the lymph nodes. Stage III pancreatic cancer has spreadto the lymph nodes near the pancreas. Cancer may have spread to nearbyorgans. Stage IV pancreatic cancer has spread to organs near thepancreas (stage IVA) or to organs far away from the pancreas (stageIVB). Stage IVA pancreatic cancer has spread to organs that are near thepancreas (such as the stomach, spleen, or colon) but has not spread todistant organs (such as the liver or lungs). Stage IVB pancreatic cancerhas spread to distant organs (such as the liver or lungs).

Selection of a Treatment Method

Identifying the presence of increased promoter methylation in BNC1 andADAMTS1, indicates that the subject likely has an invasive cancer or apre-cancerous lesion (e.g., cancer-in-situ). Further diagnostic testingmay be carried out to localize the pathology to a particular organ ororgan system. For example, endoscopic ultrasound, MRI, CT scan, PETscan, bronchoscopy, colonoscopy, esophagogastroduodenoscopy,laparoscopic surgery to localize a lesion or any other modality known inthe art.may be used to characterize the neoplasia or pre-cancerouslesion (e.g. PanIN's, or precursor lung conditions such as dysplasia,carcinoma in situ, atypical adenomatous hyperplasia or precursor coloniclesions such as adenomas). Pre-cancerous lesions are likely to besusceptible to conservative treatment methods. Conservative treatmentmethods include, for example, cancer surveillance, which involvesperiodic patient monitoring using diagnostic assays of the invention,alone or in combination, with diagnostic imaging or chemoprevention.

More aggressive neoplasias are less susceptible to conservativetreatment methods. For aggressive neoplasias, an aggressive method oftreatment should be selected. Aggressive therapeutic regimens typicallyinclude one or more of the following therapies: surgical resection,radiation therapy, and chemotherapy. After a subject is diagnosed ashaving a neoplasia (e.g., prostate cancer, lung cancer, colon cancer) amethod of treatment is selected. Where the methods of the inventionidentify a subject as having a neoplasia associated with increasedpromoter methylation in BNC1 and ADAMTS1, therapy to reduce thispromoter methylation may be selected. For example, epigenetic therapymay be selected to relieve promoter methylation in BNC1 and ADAMTS1.Such epigenetic therapy may involve the use of histone deacetylaseinhibitors or methylation inhibitors. Exemplary agents include, but arenot limited to, entinostat, SAHA (suberoylanilide hydroxamic acid),depsipeptide (Fujisawa Pharmaceuticals), azocytidine, deazocytidine,romidepsin (Istodax), Vorinostat, polyamine analogues, zebularine andother novel emerging drugs targeting methylation, histone changes or theentire polycomb complex. Epigenetic therapy may be combined with moreconventional therapies.

Patient Monitoring

The diagnostic methods of the invention are also useful for monitoringthe course of a pre-cancerous lesion in a patient or for assessing theefficacy of a therapeutic regimen. In one embodiment, the diagnosticmethods of the invention are used periodically to monitor themethylation levels of BNC1 and/or ADAMTS1. In one example, the neoplasiais characterized using a diagnostic assay of the invention prior toadministering therapy. This assay provides a baseline that describes themethylation level of one or more promoters or the methylation profile ofthe neoplasia prior to treatment. Additional diagnostic assays areadministered during the course of therapy to monitor the efficacy of aselected therapeutic regimen. A therapy is identified as efficaciouswhen a diagnostic assay of the invention detects a decrease inmethylation levels at one or more promoters relative to the baselinelevel of methylation.

Kits

The invention also provides kits for the diagnosis or monitoring of aneoplasia in a biological sample obtained from a subject. In variousembodiments, the kit includes at least one primer or probe whose bindingdistinguishes between a methylated and an unmethylated BNC1 and ADAMTS1promoter sequence, together with instructions for using the primer orprobe to identify a neoplasia. In another embodiment, the kit furthercomprises a pair of primers suitable for use in a polymerase chainreaction (PCR). In yet another embodiment, the kit further comprises adetectable probe. In yet another embodiment, the kit further comprises apair of primers capable of binding to and amplifying a referencesequence. In yet other embodiments, the kit comprises a sterilecontainer which contains the primer or probe; such containers can beboxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, orother suitable container form known in the art. Such containers can bemade of plastic, glass, laminated paper, metal foil, or other materialssuitable for holding nucleic acids. The instructions will generallyinclude information about the use of the primers or probes describedherein and their use in diagnosing a neoplasia. Preferably, the kitfurther comprises any one or more of the reagents described in thediagnostic assays described herein. In other embodiments, theinstructions include at least one of the following: description of theprimer or probe; methods for using the enclosed materials for thediagnosis of a neoplasia; precautions; warnings; indications; clinicalor research studies; and/or references. The instructions may be printeddirectly on the container (when present), or as a label applied to thecontainer, or as a separate sheet, pamphlet, card, or folder supplied inor with the container.

The following examples are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1: Identification of Cancer-Specific PromoterMethylation Candidate Genes in Pancreas Hypermethylome

In order to identify new DNA methylation biomarkers for pancreaticcancer, previously established whole human transcriptome microarrayscreening was used to identify genes silenced by promoterhypermethylation. To identify global hypermethylation-dependent geneexpression changes in pancreatic cancer, a genome-wide expressionarray-based approach was performed in four different human pancreaticcancer cell lines (PL45, MIA-PaCa2, Panc1, and Capan1) with apharmacologic strategy using 5-aza-deoxycycytidine and Trichostatin A(TSA) using standard array-based methodology (e.g. Agilent Technologies44K). As seen in FIG. 1A, a total of 1,427 unique genes were initiallyidentified in four cell lines, which met the criteria for candidatehypermethylated genes in the pancreatic cancer cell lines.

In order to identify genes that only showed cancer specific methylation,the following experimental validation criteria was used: 1) the gene wasexpressed in normal pancreas tissue, 2) the gene displayed no/lowmethylation in normal pancreatic tissues (cancer-free tissues), and 3)the gene was frequently methylated in pancreas cancer cell lines, andmethylation was also seen in primary tumors. Based on these criteria, 8genes (TFPI2, ASCL2, BNC1, TWIST1, BNIP3, ADAMTS1, PNMT, and EVL) wereidentified that displayed cancer-specific methylation in pancreaticcancers.

Example 2: BNC1 and ADAMTS1—DNA Methylation Biomarkers for EarlyDetection of Pancreatic Cancer

Next, the methylation status of these 8 genes (TFPI2, ASCL2, BNC1,TWIST1, BNIP3, ADAMTS1, PNMT, and EVL) was examined in a large series ofprimary pancreatic tumor samples (n=143; Stages 1-4) using methylationspecific PCR (see Table 1).

TABLE 1 Clinical information of pancreas cancer patient primary tumorsamples in this study Pancreas Primary tumors Normal PanINs Stage 1Stage 2 Stage 3 Stage 4 (n = 4) (N = 20) (N = 38) (N = 78) (N = 5) (N =2) Median Survival Not Met 43.0 18.0 14.1 19.6 (months) Gender Male3(75%) 9 (45%)  9(23.7%) 44 (57%) 1 (20%) 2 (100%) Femal 1925%) 11(55%)   29(76.3%) 34 (43%) 4 (80%) 0.0% Grade Well- 9 (45%) 3 (7.9%)  4(5.1%) 1 (20%) 0 (0%)  Moderately 8 (40%) 23 (60.5%) 41 (52%) 4 (40%) 1(50%) Poorly 3 (15%) 12 (31.6%)   33 (42.9%) 4 (40%) 1 (50%)

As shown in FIG. 1B, the most frequently methylated gene was BNC1(90.5%), followed by ADAMTS1 (66.7%), TWIST1 (66.7%), ASCL2 (53.79%),BNIP3 (49%), TFPI2 (53.7%) EVL (46.3%), and PNMT (26.5%). Interestingly,two of the genes that demonstrated frequent methylation in this cohortof primary pancreatic cancer samples, BNC1 (90.5%) and ADAMTS1 (66.7%),also showed frequent methylation in the precursor lesions of pancreaticcancer, pancreatic intraepithelial neoplasia (PanIN) lesion.Specifically, BNC1 methylation frequency was 70% (n=14/20) and ADAMTS1methylation frequency was 25% (n=5/20).

The ability to identify high grade precursor lesions such as PanIN3 (orpancreatic carcinoma in situ) represents one of the best means forachieving early detection of pancreatic cancer, and facilitating theimplementation of chemoprevention strategies (see FIG. 1C). BNC1 wasmethylated in PanIN 1 (56%), PanIN 2 (75%), and PanIN 3 (100%) duringPanIN progression. This data indicated that DNA methylation of BNC1 geneis an early event in pancreatic carcinogenesis and that the prevalenceof methylation increases with pancreatic cancer progression.

This genome-wide DNA hypermethylome screening is based on apharmacological approach using 5-deoxy-azacytidineand TSA, which hasshown an excellent correlation between promoter DNA methylation andinhibition of gene expression and re-expression after treatment with aDNA demethylating drug, such as 5-deoxy-azacytidine, in prior studies onbreast and colorectal cancer. Here, promoter methylation of BNC1 andADAMTS1 was tested, and correlated with a lack of gene expression inpancreatic cancer cell lines (Panc1, MIA-PaCa2, Capan1, and PL45) usingquantitative RT-PCR (qRT-PCR) (FIG. 2A, upper panel). Both genes showedvirtually undetectable (or no/low) endogenous gene expression, andsignificant re-expression after 5-deoxy-azacytidine treatment. As shownin FIG. 2A, lower panel, treatment with a histone deacetylase inhibitor,such as TSA, resulted in minimal re-expression, except in the case ofPL45, which may be regulated both by promoter DNA methylation andhistone modifications. Promoter-associated CpG island methylation in theBNC1 promoter was detected by bisulfite sequencing analysis inpancreatic cancer cell line (Panc1), a primary pancreas cancer sample(stage 2), and normal pancreatic tissue. As shown in FIG. 2B, bisulfitesequencing analysis showed dense methylation in the pancreatic cancercell line and the primary pancreatic cancer and minimal/no methylationin normal pancreas samples consistent with the methylation specific PCRanalysis.

Example 3: Detection of DNA Methylation in Pancreatic Cancer PatientSera Using Methylation on Beads Technology

Early detection of disease results in an improved clinical outcome formost types of cancer. This is particularly relevant for pancreaticcancer, which is poorly accessible and difficult to diagnose.Consequently, 90% of patients with pancreatic cancer present withmetastatic or advanced regional disease. Based on the methylationstudies, BNC1 and ADAMTS1 have emerged as important biomarkers for theearly detection of pancreatic cancer. To determine whether BNC1 andADMTS1 promoter DNA methylation could be detected in pancreatic cancerpatient sera, a highly-sensitive nanoenabled assay, termed methylationon Beads was used, which was developed to improve sensitivity ofmethylation detection (see, e.g., FIG. 2C). To test the utility ofmethylation on beads as a tool for early detection of pancreaticcancers, serum samples from a series of pancreatic cancer patients(n=42; stages 1-4), as well as a panel of normal healthy serum samples(n=26) was tested. Sensitivity was determined based on the assumptionthat all patients with cancer would be methylated for both genes, whileall healthy normal volunteers would be unmethylated for both genes.Methylation was analyzed in 42 serum samples from pancreatic cancerpatients and 26 normal volunteers. 33 of the 42 pancreatic cancerpatients showed methylation for BNC1, while 20 of the 42 showedmethylation for ADAMTS1 (see Table 2).

Overall, for all stages included, the sensitivity for BNC1 and ADAMTS1was determined to be 78.6% and 47.6%, respectively. Sensitivity ofdetection of stage I pancreatic cancers was 90% for both genes. Amongstthe 26 normal serum samples, 3 of the normal volunteers showedmethylation for BNC1 while 2 showed methylation for ADAMTS1. Specificityof detection was determined to be 88.4% for BNC1 and 92.4% for ADAMTS1.Computed results have been presented using a 95% confidence interval andare presented in detail in Table 2. Results in the corresponding tumorwere higher for both genes, with 100% sensitivity and specificity forBNC1 and 79.2% sensitivity and 100% specificity for ADAMTS1.

Additionally, the sensitivity and specificity of BNC1 and ADAMTS1 wasalso tested in other types of cancer, including both colon cancer andlung cancer (Table 2). This data showed that BNC1 was also a usefulbiomarker for the detection of early stages of colon cancer. Forexample, BNC1 showed a sensitivity of 84.6% (n=11/13) for stage 1 coloncancers and advanced adenomas. Similarly, BNC1 also showed goodsensitivity for the detection of early stage lung cancer as stage 1 lungcancer was detected with a sensitivity of 75% (n=12/16).

TABLE 2 Sensitivity and specificity of BNC1 and ADAMTS1 genes in variouscancer patient sera samples BNC1 ADAMTS1 Sensitivity(%) Estimated 95%Estimated 95% Stage N Value CI Value CI Cancer Types: Pancreatic CancerI 10 90.0% (9/10)  90.0% (9/10)  II-IV 32 75.0% (24/32) 34.4% (11/32)Total 42 78.6% (33/42) 60-90% 47.6% (20/42) 40-70% Colon CancerAdenomas, 13 84.6% (11/13) 61.5% (8/13)  stage I II-III 15 66.7% (10/15)73.3% (11/15) Other cancer* 2 100.0% (2/2)   50.0% (1/2)  Total 30 76.7%(23/30) 70-90% 66.7% (20/30) 40-70% Lung Cancer I 16 75.0% (12/16) 62.5%(10/16) II-III 4 100.0% (4/4)   100.0% (4/4)   Total 20 80.0% (16/20)60-90% 70.0% (14/20) 50-90% Normal Specificity 26 88.4% 70-90% 92.4%70-90% CI: Confidential Interval. *other colorectal malignancies.

A subset of tissue samples from patients diagnosed with non-cancerouspancreatic diseases, such as pancreatitis, was collected and analyzed.Patients with chronic pancreatitis maybe at increased risk forpancreatic cancer and amongst patients with pancreatic cancer, 5% ormore of patients have underlying chronic pancreatitis. Chronicinflammatory conditions likely increases the frequency of methylation asa field defect which may then increase risk of subsequent malignancy.The methylation of BNC1 and ADAMTS1 DNA was compared between differentconditions (normal, pancreatitis, PanIN, and invasive cancers) usingquantitative methylation specific PCR analysis. As shown in FIG. 3, BNC1and ADAMTS1 showed statistically increased frequency of methylation whencomparing normal pancreas tissues and invasive cancers (p<0.0001; bothBNC1 and ADAMTS1), as well as chronic pancreatitis and invasive cancers(p<0.0001, both BNC1 and ADAMTS1). There was a low frequency of BNC1 andADAMTS1 methylation present in non-cancerous disease, such aspancreatitis. More interestingly, there was a significant quantitativedifference between PanINs and invasive cancers on both genes (p<0.001both BNC1 and ADAMTS1). However, BNC1 methylation could be detected inthe earliest stages of pancreatic carcinogenesis such as PanIN's,compared to ADAMTS1 methylation (see FIG. 3), indicating that BNC1methylation is a very early event, and that ADAMTS1 methylation is alater event during pancreas cancer development.

Example 4: Tumor Suppressive Effects of BNC1 Gene Over-Expression inPancreatic Cancer Cells

According to the methylation data in pancreatic cancer patients, BNC1 isa useful DNA methylation biomarker in pancreatic cancer patients. Todetermine whether BNC1 played a role as a tumor suppressor in pancreaticcancer cells, in vitro colony formation assays were performed todetermine the effects of full-length BNC1 transfected into Panc1 andMIA-PaCa2 cells lacking BNC1 expression. Compared with control cellstransfected with empty vector, over-expression of a full-length BNC1gene induced a nearly 2.2-fold (Panc1) and 9-fold (MIA-PaCa2) reductionof G418-resistant colonies (see FIG. 4A). In addition, there was a 75%(Panc1) and 81% (MIA-PaCa2) decrease in cell proliferation as measuredby [³H] thymidine activity (FIGS. 4B and 4C; left and middle panel).However, overexpression of BNC1 gene had no effect on Panc1 andMIA-PaCa2 cells migration and invasion through matrigel-coated transwellmembranes compared with control cells transfected with empty vector(FIGS. 4B and 4C; right panel). Taken together, these data indicatedthat BNC1 gene has tumor-suppressive effects in human pancreatic cancercells.

In this study, genome-wide gene expression profiling using apharmacological approach (5-deoxy-azacytidine and TSA) was used inpancreatic cancer cell lines, a platform which has been used to identifyan early detection marker for colorectal cancer. Defining the DNAhypermethylome has been useful as it has allowed the identification ofnot only novel DNA methylation biomarker candidates, but also of tumorsuppressor gene candidates in many types of cancers (colon, breast,ovarian, etc). The identified DNA methylation biomarkers will facilitatediagnostics, and also contribute to therapeutics as predictors ofresponse to therapy.

The poor accessibility of the pancreas along with the late presentationof symptoms has thwarted attempts at timely detection of malignancy, andcontributed to the high mortality rates of pancreatic cancer, which isthe fourth leading cause of cancer death in both men and women.Therefore, the development of cancer biomarkers of pancreatic cancer isthe best hope for early detection.

As reported herein above, the promoter methylation of BNC1 and ADAMTS1is an excellent early detection biomarker for pancreatic cancer. BNC1and ADAMTS1 have not been described as a DNA methylation biomarker inpancreatic cancers, although they have been reported methylated in lungcancers.

Screening with a nanobased high sensitive technology allowed thesensitive and specific detection of pancreatic cancer in its earlieststages. Using BNC1 and ADAMTS1, provided for the detection of very earlystages of pancreatic carcinoma, with an overall sensitivity of 83.7%(95% CI; 74.2-90.3%) and a specificity of 84.6% (95% CI; 64.3-95.0%).Significantly, the sensitivity achieved in this study is higher thanpreviously reported for serum hypermethylation markers. The presentinvention provides a cost-effective approach for screening individualsidentified as having risk factors for pancreatic cancer in the generalpopulation. Additionally, the nano-based methylation on beads detectionmethod significantly reduced the quantity of serum necessary foranalysis. The invention provides a paradigm for widespread screening forpancreatic cancers using a simple blood test.

In conclusion, this is the first study to describe the utility of BNC1and ADAMTS1 promoter methylation as biomarkers in pancreatic cancerpatient serum using nanoparticle-enabled technology. In addition, thisis the first report to demonstrate a tumor suppressor role for BNC1 inpancreatic cancer. These data indicate that BNC1 promoter methylation isuseful as a sensitive and specific noninvasive pre-selection modalityfor diagnosing subjects as having cancer and identifying individuals atrisk for pancreatic cancer.

The results presented hereinabove were carried out using the followingmethods and materials.

Gene Expression Microarray Analysis

Total RNA was harvested from log phase cells using standard methods(e.g. with TRIzol (Invitrogen™) and the RNeasy kit (Qiagen™) accordingto the manufacturer's instructions), including a DNase digestion step.RNA was quantified using a spectrophotometer (e.g. the NanoDrop™ ND-100(http://www.nanodrop.com/)) followed by quality assessment with amicrofluidics analysis platform (e.g. the Agilent™ 2100 Bioanalyzer(Agilent Technologies, http://www.agilent.com/). RNA concentrations forindividual samples were greater than 200 ng/ll, with 28S/18S ratiosgreater than 2.2 and RNA integrity of 10 (10 scored as the highest).Sample amplification and labeling procedures were carried out usingstandard methods (e.g. Low RNA Input Fluorescent Linear AmplificationKit (Agilent Technologies™) according to the manufacturer'sinstructions). The labeled cRNA was purified using spin columns (e.g.the RNeasy mini kit (Qiagen™)) and quantified. RNA spike-in controls(Agilent Technologies™) were added to RNA samples before amplification.Samples (0.751 g) labeled with Cy3 or Cy5 were mixed with controltargets (Agilent Technologies™), assembled on Oligo Microarray,hybridized, and processed according to the Agilent microarray protocol.Scanning was performed with a microarray scanner (Agilent™ G2565BA,using recommended settings). Data analysis was conducted as previouslyreported (Schuebel K, Chen W, Cope L, Glöckner S C, Suzuki H, et al.,Comparing the DNA hypermethylome with gene mutations in human colorectalcancer. 2007, PLOS Genetics, 3(9):1709-1723.

Patient Samples and Study Population

Patient samples were prepared from formalin-fixed, paraffin-embedded(FFPE) tissue samples from patients with pancreatic cancer procured fromthe pathology archives of the Johns Hopkins Hospital in accordance withall rules and regulations of the Institutional Review Board (IRB) and asper HIPAA compliance. A total of 167 patients were analyzed in thecurrent study. The Johns Hopkins cohort consisted of 143 tissue samplesfrom pancreatic cancer patients with stage 1 through stage 4 disease whounderwent primary surgery and other treatment for pancreatic cancer atthe JHH from 1998 to 2009 (Median follow up of 6.4 years). Patients inthis cohort were similar by stage with respect to gender, proportion ofcases with lymphovascular invasion, and pathologic grade (Table 1). Anadditional 20 FFPE tissue samples were obtained from patients who hadundergone surgical resection but diagnosed with precancerous lesions,pancreatic intraepithelial neoplasias (PanINs). Finally, 4 FFPE tissuesamples were obtained from pathology archives from patients who hadundergone pancreatic resection with no abnormality noted on gross ormicroscopic examination.

Pancreatic Cancer Patient Serum Samples

Patient serum samples were obtained from individuals with pancreaticcancer prior to undergoing surgical treatment at the JHH after obtaininginformed consent from 2007 to 2009. Matching tumor samples were drawnfrom the pathology archives of the Johns Hopkins Hospital in accordancewith all rules and regulations of the Institutional Review Board (IRB)and as per HIPAA compliance. A total of 36 serum samples were tested, 23of which had matching FFPE tissue available. Patient serum samples werealso obtained from individuals with pancreas (42 samples, Stages 1-4),colon (30 samples, Stages 1-3 and adenomas) and lung cancers (20samples, Stages 1-3) prior to undergoing surgical treatment at the JHHafter obtaining informed consent. Additionally, 26 serum samples wereobtained from normal, healthy volunteers to serve as controls.

Cell Culture and Treatment

Cancer cell lines (Pancreas cancer cell lines; Panc1, MIA-PaCa2, PL45,Capan1, Bxpc3, Capan2, PK9, Aspc1, PK8, and PL3) were obtained from ATCCand cultured in appropriate media and under conditions described byATCC, with media obtained from InVitrogen™, supplemented with 10% fetalbovine serum (Gemini Bio-Products) and 1% penicillin/streptomycin(InVitrogen™). DKO cells (HCT116 colon cancer cells with geneticdisruption of DNMT1 and DNMT3b) were cultured as described previously(Rhee et al., 2002, Nature).

DNA Methylation Analyses and RT-PCR for Expression Analysis

Primer pairs were preferentially designed near the putativetranscriptional start site (TSS) in the 5′ CpG islands of the genes.Primer sequences for methylation specific PCR analysis were designedusing MSPPrimer (http://www.mspprimer.org). All primer sequences arelisted in

TABLE 3 For expression studies using RT-PCR, primers weredesigned using the open access program Primer3(http://frodo.wi.mit.edu/cgi-bin/ primer3/primer3_www.cgi).Table 3: Primers used for MSP, RT-PCR, Bisulfite sequencing analysesGenes Primers S(5-3) AS(5-3) ADAMTS1 Unmeth tTATTGtAAAGttA aaaaCTaAaaCAAGGGTGtGtTAttGG CaCaaAaATTaaT AtG aCCTaaCa Meth ttAGGGTGCGtTAtTaAaaCAACGCGa CGGAC AaATTaaTaCCTa aCG RT tcgaagtgaaacag gccgctgtacctccggaac aagacaa ASCL2 Unmeth tGtTAGGGGAtAGt CaTACaCaTaaCa GtGtttAGtttAGTCaTaaCTCACaaa tGtG aAaCATCa Meth GAtAGCGCGtttAG TaaCGCGTaaCTC tttAGTCGCACGaaaAaCATCG BNC1 Unmeth TTtGtttTTttttG aaAaACCTCaCCa GGAGAGGtAAAtAtaCaaCCaaCa tGAtAtG Meth GtttTTtttCGGGA CCGaCGaCCGaCG GAGGtAAAtAtCGA tACRT acaaaagcctggcc tcgccccaaatga tcatct tatgaaa Bisulfite AATtATtTttTGAGCCCAAaCRCCCAa Sequencing AAGAGYGttAGAGA aCTaC AtT BNIP3 UnmethttAtGttttTGtGt aCTCCCaaACTaA AtGtGtAGGttttA aCaaAaCCCCa AGTtGtG MethGtACGCGtAGGttt CCGaACTaAaCGa tAAGTCGC AaCCCCG EVL Unmeth GTGTGTTTTTTTTTACCACCAAAAAAT TGAGGATTTGGAGT AAAAAAACAAAAA TGTTTG ACAAACCA MethGAGGATTCGGAGTC CCGAAAAATAAAA GTTC AAACGAAAAACGA ACCG PNMT UnmethTGGGGGAtGATTGt CaCCaAaCCaCCC tGtTGtAGttG aaaaCCATaTaCa MethGGGGACGATTGtCG AaCCGCCCGaaaC tTGtAGtC CATaTaCG TFPI2 UnmethCCCACATAAAACAA TGGTTTGTTGGGT ACACCCAAACCA AAGGTGTTTG Meth GTTCGTTGGGTAAGCATAAAACGAACA GCGTTC CCCGAACCG TWIST1 Unmeth tAGAtAttTtGtGGaCaaaAaaaaaAa GtTtTGtAGtAttG aaaACaaTaTaaA GtAttG TaaCCCCa MethCGGGtTtTGtAGtA CGAaAaaaaaAaa tCGGtAtC aaACGaTaTaaAT aaCCCCG

For methylation-specific PCR analysis, DNA was extracted using thestandard phenol-chloroform extraction method. Bisulfite modification ofgenomic DNA was carried out using standard protocols (e.g. with the EZDNA methylation Kit (Zymo Research™)). Methylation analysis of CAN gene(Cancer gene, as defined by Wood L D, Parsons D W, Jones S et al., Thegenomic landscapes of human breast and colorectal cancers, Science.2007, 318(5853):1108-1113) promoters was performed using MSP primerpairs located close to the putative transcriptional start site in the 5′CpG island with 2 μl of bisulfite-treated DNA as template and JumpStartRed Taq DNA Polymerase (Sigma™) for amplification as previouslydescribed (Herman J G, Graff J R, Myohanen S, Nelkin B D, Baylin S B.,Methylation-specific PCR: a novel PCR assay for methylation status ofCpG islands. Proc Natl Acad Sci USA. 1996 Sep. 3; 93(18):9821-9826).

Total RNA was extracted from cell lines using standard methods (e.g. theRNeasy Mini Kit (Qiagen™)), and treated with DNase. For reversetranscription (RT) reaction, 1 μg of total RNA was subjected to thefirst strand cDNA synthesis using standard cDNA synthesis protocols(e.g. Superscript III first strand cDNA synthesis kit (InVitrogen™) usedaccording to the manufacturer's instructions). Expression analysis wasperformed by RT-PCR using 1 μl of cDNA as template and standardamplification protocols (e.g. JumpStart Red Taq DNA Polymerase(Sigma™)).

Quantitative Methylation-Specific PCR Using Real-Time PCR

Sodium bisulfite modification, which converts unmethylated cytosineresidues to uracil residues, was first carried out on 1 ug genomic DNAisolated from the paraffin-embedded tissue sections using standardmethylation procedures (e.g. EZ DNA methylation kit (Zymo Research Co)according to the manufacturer's instructions). For quantitativereal-time analyses, standard procedures were used. For example, thePower SYBR Green PCR kit (Applied Biosystems) was used and theamplification conditions consisted of an initial 10 minute denaturationstep at 95° C., followed by 40 cycles of denaturation at 95° C. for 15seconds and annealing and extension for 30 seconds and 60 seconds,respectively. An ABI StepOnePlusReal-Time PCR System was used (AppliedBiosystems), and for quantification the comparative cycle threshold (Ct)method was used, normalizing the Ct values for the indicated gene to theCt values of Unmethylated reaction relative to a methylated reactionsample.

Methylation on Beads (MOB) Method

Methods and buffers used for methylation on beads extraction are asdescribed previously (Bailey V J, Zhang Y, Keeley B P, Yin C, Pelosky KL, Brock M, Baylin S B, Herman J G, Wang T H. Single-tube analysis ofDNA methylation with silica superparamagnetic beads. Clin Chem.56(6):1022-1025). Briefly, 50 μL of Protease K was added into the bottomof an eppendorf tube, and 200 μL methylation on beads Binding Buffer 1and 100 μL Lysing Buffer to 200 μL was added to the serum sample. Thesample was incubated at 55° C. for 15 minutes (cell lines, whole blood),and 200 μL 100% Isopropyl Alcohol was added, and the sample was mixed.35 μL of Magnetic Beads was then added to the sample and mixed. The tubewas then placed on the magnetic holder to capture the beads, and thesupernatant was discarded. The tube was removed from the magneticholder, and the beads were washed with 500 μL of Wash Buffer 1. Thebeads were once again captured on the magnetic holder, and then washed asecond time in 500 μL of Wash buffer 2. This step was then repeated onetime, and the beads were again captured, and the supernatant wasdiscarded. 50 μL of Elution Buffer was added to the eppendorf tubecontaining the washed beads. To denature the DNA for bisulfitetreatment, 6 μL 2M NaOH was added to the tube, mixed, and incubated at70° C. for 10-15 minutes. 12 μL of freshly prepared hydroquinonesolution was then added and mixed. 200 μL of prepared NaBisulfitesolution was added and mixed. The NaBisulfite solution was vortexed toinsure that it was fully saturated. The sample was incubated at 65° C.for 4-6 hours, and then 120 μL of 10M NaOH was added, mixed, andincubated at 50° C. for an additional 10 minutes. 350 μL of methylationon beads Binding Buffer 2, 100 μL of IPA was added to the sample andmixed. The beads were then captured and washed once in Wash Buffer 1 andtwice in Wash Buffer 2 as described above. The beads were then capturedand the supernatant discarded. The DNA was then eluted from the magneticbeads with 100 μL of PCR buffer. The sample was then divided as desiredfor subsequent PCR amplification.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A method for detecting or characterizing a neoplasia in a biologicsample of a subject, the method comprising detecting the methylation ofa BNC1 and/or ADAMTS1 gene, wherein detection of methylation detects orcharacterizes the presence of a neoplasia in the sample.
 2. The methodof claim 1, wherein the method detects an increase in methylationrelative to a reference.
 3. The method of claim 1, wherein the methoddetects promoter methylation or methylation of exon
 1. 4. The method ofclaim 1, wherein the neoplasia is a cancer selected from the groupconsisting of pancreatic cancer, gastrointestinal cancer, lung cancer,colon cancer, duodenal cancer, colorectal carcinoma, neuroendocrinecarcinoma, cholangiocarcinomas, and ampullary tumors.
 5. The method ofclaim 1, wherein the biologic sample is a tissue or biologic fluidsample.
 6. The method of claim 1, wherein the biologic fluid is selectedfrom the group consisting of blood, serum, plasma, urine, pancreaticjuice, pancreatic cyst fluid, or lung lavage.
 7. The method of claim 2,wherein the reference is the level of methylation present at thepromoter in a control sample.
 8. The method of claim 7, wherein thecontrol sample is derived from a healthy subject.
 9. The method of claim1, wherein the methylation is detected by quantitativemethylation-specific PCR (QMSP). 10-20. (canceled)
 21. A method fordetecting or characterizing pancreatic cancer in a serum or plasmasample derived from a subject, the method comprising detecting themethylation of BNC1 and ADAMTS1, wherein detection of methylationdetects or characterizes pancreatic cancer in the subject.
 22. Themethod of claim 21, wherein the method detects an increase inmethylation relative to a reference.
 23. The method of claim 17, furthercomprising imaging the subject, and localizing the cancer.
 24. Themethod of claim 21, further comprising detecting an alteration in thesequence or expression of a Brca1, Brca2, p16, K-ras, APC, PalB2, and/orDPC4 gene or polypeptide relative to a reference. 25-27. (canceled) 28.A method of monitoring a subject diagnosed as having a neoplasia, themethod comprising detecting an alteration in promoter methylation levelin a BNC1 and/or ADAMTS1 gene in a subject sample relative to areference, wherein an altered level indicates an altered severity ofneoplasia in the subject; or A method for selecting a treatment for asubject diagnosed as having a neoplasia, the method comprising detectingmethylation of a BNC1 and/or ADAMTS1 gene, wherein detection ofmethylation indicates that epigenetic therapy should be selected fortreatment of said subject. 29-32. (canceled)
 33. The method of claim 1,wherein the subject is a human patient.
 34. The method of claim 1,wherein the methylation is quantified by methylation on beads orquantitative methylation-specific PCR. 35-36. (canceled)
 37. A kit forthe analysis of promoter methylation, the kit comprising at least oneprimer capable of distinguishing between methylated and unmethylatedBNC1 and ADAMTS1 promoter sequences.
 38. The kit of claim 37, furthercomprising a pair of primers for amplifying the promoter sequence of areference gene.
 39. The kit of claim 37, further comprising a detectableprobe, wherein the probe is capable of binding to the promoter sequence.40. The kit of claim 39, wherein the probe is detected by fluorescence,by autoradiography, by an immunoassay, by an enzymatic assay, or by acolorimetric assay.